Intermediates for preparing neuraminidase inhibitor conjugates

ABSTRACT

Compounds of formula (I), methods for their preparation and their use in the manufacture of neuraminidase inhibitor conjugates. Wherein R represents as carboxylic acid protecting group; P 1  and P 2  can be the same or different and are selected from amine protecting groups; P 3  represents a protecting group for 1, 2 diols; and LG represents a leaving group.

The present invention relates to novel compounds, methods for theirpreparation and their use in the manufacture of neuraminidase inhibitorconjugates.

Dimeric compounds and their use as neuraminidase inhibitors have beendisclosed in WO00/55149. Polymeric compounds and their use asneuraminidase inhibitors have been disclosed in WO98/21243. InWO00/55149, it was shown that when two neuraminidase binding compoundsare suitably linked together through a region of the molecule that isnot involved in binding to the active site, the resultant dimers haveenhanced anti-viral activity. Eur. J. Med. Chem 34 (1999) 563-574discloses the synthesis and influenza virus sialidase inhibitory actionof an analogue series of 4-guanidino-Neu5Ac2en (zanamivir) modified inthe glycerol side chain.

In WO00/55149, compound 7 is described as a useful precursor to certaindimeric neuraminidase inhibitors.

We have found that in a first aspect the invention provides compounds offormula (I):

wherein R represents a carboxylic acid protecting group;

P₁ and P₂ can be the same or different and are selected from amineprotecting groups; P₃ represents a protecting group for 1,2 diols; and

LG represents a leaving group, for example, para-nitrophenol or aderivative thereof, halide, imidazole or N-hydroxysuccinimide.

Preferably R is C₁₋₆ alkyl, diphenylmethane or an appropriate protectinggroup selected by one skilled in the art from common carboxylic acidprotecting groups such as those listed in “Protective Groups in OrganicSynthesis,” TW Greene and PGM Wuts 1999 (3^(rd) edition), Wiley.

When used herein an alkyl group can be straight, branched or cyclic forexample methyl, ethyl, propyl, isopropyl, butyl, t-butyl, pentyl, hexylor cyclohexyl, preferably methyl or t-butyl.

Common amine protecting groups are as those listed in “Protective Groupsin Organic Synthesis,” TW Greene and PGM Wuts 1999 (3^(rd) edition),Wiley, preferably a t-butoxycarbonyl (Boc) group.

Protecting groups for 1,2 diols are CO (a cyclic carbonate) or CHMe (amethyl acetal) or an appropriate protecting group selected by oneskilled in the art from common 1,2 diol protecting groups such as thoselisted in “Protective Groups in Organic Synthesis,” TW Greene and PGMWuts 1999 (3^(rd) edition), Wiley. Preferably P₃ represents CO or CHMe.

Other leaving groups will be known to the person skilled in the art forthe preparation of carbamates.

Even more preferably R is methyl or diphenylmethane, P₁ and P₂ are Boc,P₃ is CO and LG is para-nitrophenol.

Compounds of the present invention offer a significant advantage in therapid preparation of large numbers of neuraminidase inhibitorconjugates, specifically those disclosed in WO 00/55149. Compounds ofthe present invention provide a common intermediate from which a largenumber of neuraminidase inhibitor conjugates can be prepared usingdifferent “linking groups” many of which are commercially available.Using a common intermediate allows flexibility and the ability toproduce large numbers of compound quickly.

Compounds of formula (I) may be useful in the preparation of compoundlibraries comprising at least 2, e.g. 5 to 1000, compounds, preferably10 to 100 compounds.

Compound libraries may be prepared by “split and mix” approach or bymultiple parallel synthesis using either solution phase or solid phasechemistry, by process known in the art.

A second aspect of the invention is the use of compounds of formula (I)in the preparation of neuraminidase inhibitor conjugates, specificallythose disclosed in WO 00/55149.

A third aspect of the invention is the process for the preparation ofneuraminidase inhibitor conjugates, specifically those disclosedWO00/55149 comprising the use of compounds of formula (I).

A further aspect of the invention is neuraminidase inhibitor conjugates,specifically those disclosed in WO00/55149, prepared using compounds offormula (I).

WO00/55149 and WO98/21243 are incorporated by reference as if eachindividual publication were specifically and individually indicated tobe incorporated by reference herein as though fully set forth.Specifically the generic formula of the neuraminidase inhibitorconjugates are incorporated herein.

Compounds of formula (I) can be prepared by reaction of compounds offormula (III);

wherein P₁, P₂, P₃ and R are as described for compounds of formula (I),with compounds of formula (II);

wherein each LG is independently as described for compounds of formula(I), in a solvent, and a base.

Preferably the base used is a tertiary amine, for exampledimethylaminopyridine (DMAP), 4-pyrrolidinopyridine or1,8-diazabicyclo[5.4.0]undec-7-ene, more preferably DMAP.

Preferably at least two equivalents of base to compound of formula (III)are used.

Preferably the solvent is pyridine or a pyridine type solvent.

Preferably the reaction should be carried out in the absence of water,for example by azetroping the starting materials, or drying in an ovenprior to carrying out the reaction.

For example compounds of formula (II) may be symmetrical orunsymmetrical e.g. p-nitrophenylchloroformate.

Compounds of formula (III) can be prepared by reaction of compounds offormula (IV);

wherein P₁, P₂ and R are as described for compounds of formula (1), withcarbonyldiimidazole (CDI) or phosgene or other phosgene equivalents.

Compounds of formula (IV) wherein R is diphenylmethane are known in theliterature, J Med Chem 1998, 41, 787-797.

Neuraminidase inhibitor conjugates of formula (V);

may be prepared by reacting compounds of formula (I) with compounds offormula (VI);

in solvent, for example pyridine, and in the presence of base, forexample DMAP, followed if necessary by deprotection.

Methods of deprotecting the amine and ester groups will be well known tothe person skilled in the art.

When used herein halide represents a fluoro, chloro, bromo or iodogroup.

Compounds of formula (V) can be tested for neuraminidase activity bymethods known in the art for example by plaque assays, Hayden et al.(Antimicrobial. Agents Chemother., 1980, 17, 865).

The invention will now be described in detail by way of reference to thefollowing non-limiting examples.

Examples 1 and 2 disclose the preparation of compounds of formula (I).Example 3 describes the preparation of a neuraminidase inhibitorconjugate of formula (V).

Abbreviations used herein are

-   DPM-diphenylmethane-   SPE-solid phase extraction.-   DMAP-4-dimethylaminopyridine-   BOC-t-butoxycarbonyl-   EtOAc-ethyl acetate-   DCM-dichloromethane-   THF-tetrahydrofuran-   CDI-1,1′-carbonyldiimidazole-   LC/MS liquid chromatography mass spectrometry.

EXAMPLE 1

Intermediate 1 Benzhydryl(2R,3R,4S)-3-(acetylamino)-4-({[(tert-butoxycarbonyl)amino][(tert-butoxycarbonyl)imino]methyl}amino)-2-{(S)-hydroxy[(4R)-2-oxo-1,3-dioxolan-4-yl]methyl}-3,4-dihydro-2H-pyran-6-carboxylate

Benzhydryl(2R,3R,4S)-3-(acetylamino)-4-({(E)-[(tert-butoxycarbonyl)amino][(tert-butoxycarbonyl)imino]methyl}amino)-2-[(1R,2R)-1,2,3-trihydroxypropyl]-3,4-dihydro-21′-pyran-6-carboxylate(see J. Med. Chem. 1998, 41, 787-797) (12.38 g; 17.7 mmoles) wasdissolved in dry acetonitrile (130 mil) under nitrogen at roomtemperature. The solution was stirred and 1,1′-carbonyldiimidazole (2.87g; 17.7 mmoles) was added. After 16 hours LC/MS showed the presence ofstarting triol so further 1,1′-carbonyldiimidazole (total of 0.493 g; 3mmoles) was added. After a few hours LC/MS showed no triol present. Thesolvent was evaporated and the residue flash columned on silica, elutingwith 1:1 ethyl acetate/40-60 petroleum ether. Fractions containingwanted product were evaporated then taken up in dichloromethane, driedwith sodium sulphate, filtered and evaporated to give Intermediate 1 asan off white solid (11.05 g; 86%).

LC/MS (Blue method) MH⁺=725, T_(ret)=4.09 minutes.

EXAMPLE 1

Benzhydryl(2R,3R,4S)-3-(acetylamino)-4-({(E)-[(tert-butoxycarbonyl)amino][(tert-butoxycarbonyl)imino]methyl}amino)-2-{(S)-{[(4-nitrophenoxy)carbonyl]oxy}[(4R)-2-oxo-1,3-dioxolan-4-yl]methyl}-3,4-dihydro-2H-pyran-6-carboxylate

A solution of benzhydryl(2R,3R,4S)-3-(acetylamino)4({[(tert-butoxycarbonyl)amino][(tert-butoxycarbonyl)imino]methyl}amino)-2-{(S)-hydroxy[(4R)-2-oxo-1,3-dioxolan-4-yl]methyl)-3,4-dihydro-2H-pyran-6-carboxylate(Intermediate 1)(143 mg, 0.197 mmol) in dry pyridine (3 ml) containing4-dimethylaminopyridine (120 mg, 0.982 mmol) was treated with4-nitrophenylchloroformate (199 mg, 0.987 mmol) at 22° C. The mixturewas stirred at 22° C. For 17 h, then the pyridine removed in vacuo. Theresidue was purified by SPE chromatography (5 g cartridge) eluting withcyclohexane-ethyl acetate (4:1-2:1) to afford Example 1 as a pale yellowgum (99 mg, 56%).

NMR δ(CDCl₃) 11.30 (1Hbrd, NH), 8.62 (1H brd, NH), 8.23 (2H, AA′BB′,aromatic CH's), 7.52 (2H, AA′BB′, aromatic CH's), 7.43-7.30 (10 Hm,aromatic CH's), 6.95(1Hs, CH), 6.76 (1H brd, NH), 6.05 (1Hd, ═CH), 5.56(1Hdd, CH), 5.22 (1Hdt, CH), 5.00 (1Hdt, CH), 4.72 (1Hdd, CH), 4.59(1Hdd, CH), 4.48 (1Hq, CH), 4.25 (1Hdd, CH), 1.92 (3Hs, CH3), 1.48 (9Hs,tert butyl), and 1.43 (9Hs, tert butyl).

LC/MS R₁=4.19 min. (MH⁺=890, MH⁻=888)

EXAMPLE 2

Intermediate 2 Methyl(2R,3R,4S)-3-(acetylamino)-4-amino-2-[(1R,2R)-1,2,3-trihydroxypropyl]-3,4-dihydro-2H-pyran-6-carboxylatehydrochloride

Acetyl chloride (75 ml; 1.05 mole) was added drop-wise with stirring tomethanol (7500 ml) at 0-5° C. under nitrogen. The mixture was stirred atthis temperature for a further 15 minutes then held at approximately 10°C. as(2R,3R,4S)-3-(acetylamino)₄-amino-2-[(1R,2R)-1,2,3-trihydroxypropyl]-3,4-dihydro-2H-pyran-6-carboxylicacid trihydrate (see J. Med. Chem. 1998, 41, 787-797) (250 g; 726mmoles) was added in portions. The mixture was stirred at approximately60° C. for 5 hours then cooled to approximately 20° C. and stirred atthis temperature overnight. The solvent was removed and the residuetwice evaporated down again with methanol (2×500 ml) to give a mixtureof a foam and gum. This was re-dissolved in methanol (˜1000 ml),evaporated and the residue then triturated with DCM and re-evaporated.The trituration DCM process was repeated. The residue was driedovernight in a vacuum-oven at approximately 30° C., crushed and thendried overnight again to give Intermediate 2 as a white powder (264.2g).

LC/MS (Orange Method) MH⁺305, T_(ret)=0.54 minutes.

Intermediate 3 Methyl(2R,3R,4S)-3-(acetylamino)-4-({[(tert-butoxycarbonyl)amino][(tert-butoxycarbonyl)imino]methyl} amino)-2-[(1R,2R}1,2,3-trihydroxypropyl]-3,4-dihydro-2H-pyran-6-carboxylate

The amino ester hydrochloride Intermediate 2 (211.6 g; 0.62 mole) wasadded portion-wise to methanol (2100 ml) stirring under nitrogen in a 10litre reactor to give a pale brown solution. THF (2100 ml) was added.Triethylamine (86.5 ml; 0.62 mole) was added drop-wise with stirring andthen a solution of N,N′-bis-t-butyloxycarbonyl-1-guanylpyrazole (201.3g; 0.649 mmole) in THF (2100 ml) was added drop-wise, fairly quickly,maintaining the reaction temperature at approximately 22° C. The mixturewas stirred under nitrogen at approximately 22° C. for 45 hours thenfiltered to remove a small amount of solid and the filtrate evaporatedto dryness. After standing overnight the gummy yellow residue wastriturated with ethyl acetate (2500 ml) by rotation on rotary evaporatorto give a fine white solid which was filtered off. The filtrate wasevaporated down and dried under high vacuum to give a foam (−333 g). Thefoam was dissolved in 3% methanol/DCM (˜700 ml) and purified on a 2.5 kgBiotage column pre-conditioned in and eluted with 3% methanol/DCM. Thepurest fractions were combined and evaporated then dried atapproximately 30° C. to give Intermediate 3 as a white solid (192.8 g;49.4% yield corrected for the presence of pyrazole). NMR showed thepresence of −54 mole % pyrazole (˜13% by weight).

LC/MS (Orange Method) MH⁺=547, T_(ret)=5.07 minutes.

Intermediate 4 methyl(2R,3R,4S)-3-(acetylamino)-4-({[(tert-butoxycarbonyl)amino][(tert-butoxycarbonyl)imino]methyl}amino)-2-((S)-hydroxy[(4R)-2-oxo-1,3-dioxolan-4-yl]methyl)-3,4-dihydro-2H-pyran-6-carboxylateIntermediate 3 (423.2 g; ca 0.77 mole) (contaminated with ˜13%pyrazole), was dissolved in dry acetonitrile (4750 ml) and stirred undernitrogen in a 10 litre reactor. CDI (135.6 g; 0.84 mole) was addedportion-wise using circulator to control the slight exotherm andmaintain the reaction temperature at approximately 22° C. The mixturewas stirred at this temperature under nitrogen overnight. After 22 hoursthe solvent was removed and the residual yellow gum was dissolved inethyl acetate (3500 ml) and returned to the reactor. The solution waswashed in the reactor twice with dilute hydrochloric acid (2×1250 ml; 1M), then once with water (1000 ml), then once with brine (800 ml). Thesolution was dried over magnesium sulphate, filtered, evaporated anddried in high vacuum to give a white foam (378 g). The foam wasdissolved in DCM (˜1000 ml) and the solution applied in two batches to a2.5 kg Biotage column preconditioned in and eluted with 1:1 hexane/ethylacetate to give, after evaporation and drying, Intermediate 4 as a whitesolid (total 292.1 g; ˜76% based on corrected amount of startingmaterial).

LC/MS (Orange Method) MH⁺=573, T_(ret)=5.85 minutes.

EXAMPLE 2

Methyl(2R,3R,4S)-3-(acetylamino)4({(E)-[(tert-butoxycarbonyl)amino][(tert-butoxycarbonyl)imino]methyl}amino)-2-{(S)-([(4-nitrophenoxy)carbonyl]oxy}[(4R)-2-oxo-1,3-dioxolan-4-yl]methyl)-3,4-dihydro-2H-pyran-6-carboxylate

A solution of methyl(2R,3R,4S)-3-(acetylamino)-4-({[(tert-butoxycarbonyl)amino][(tert-butoxycarbonyl)imino]methyl}amino)-2-{(S)-hydroxy[(4R)-2-oxo-1,3-dioxolan-4-yl]methyl}-3,4-dihydro-2H-pyran-6-carboxylate(113 mg, 0.197 mmol) in dry pyridine (3 ml) containing4-dimethylaminopyridine (120 mg, 0.982 mmol) was treated with4-nitrophenylchloroformate (199 mg, 0.987 mmol) at 22° C. The mixturewas stirred at 22° C. For 17 h, then the pyridine removed in vacuo. Theresidue was purified by SPE chromatography (5 g cartridge) eluting withcyclohexane-ethyl acetate (4:1-2: 1) to afford Example 2 as a paleyellow gum (96 mg, 66%).

NMR δ(CDCl₃) 11.3 (1Hs, NH), 8.58 (1H brd, NH), 8.26 (2H, AA′BB′,aromatic CH's), 7.56 (2H, AA′BB′, aromatic CH's), 6.82 (1H brd, NH),5.93 (1Hd, ═CH), 5.54 (1Hdd, CH), 5.20 (1Hdt, CH), 5.10 (1Hdt, CH), 4.78(2Hm, 2×CH), 4.44 (1H brq, CH), 4.28 (1Hdd, CH), 3.82 (3Hs CH3), 1.91(3Hs, CH3), and 1.48 (18Hs, 2×tert butyl).

LCMS R₁=3.87 min. (MH⁺=738, MH⁻=736)

EXAMPLE 3

The benzhydryl(2R,3R,4S)-3-(acetylamino)₄-({[(tert-butoxycarbonyl)amino][(tert-butoxycarbonyl)imino]methyl}amino)-2-{(S)hydroxy[(4R)-2-oxo-1,3-dioxolan-4-yl]methyl}-3,4-dihydro-2H-pyran-6-carboxylate(0.4 g; 0.55 mmole) was azeotroped 4 times from dry toluene and thedried solid was dissolved in molecular sieve-dried pyridine (1.6 ml).The solution was treated with 4-dimethylaminopyridine (0.17 g; 1.4mmoles). To this was added 4-nitrophenylchloroformate (0.12 g; 0.6mmole) under nitrogen. A slight exotherm occurred, the temperaturerising from 24° C. to 27° C. The mixture was stirred at room temperaturefor 3 hours after which time LC/MS showed the absence of startingmaterial and the presence of the nitrophenylcarbonate (Example 1)MH⁺=890.

To this mixture was added 4,7,10-trioxa-1,13-tridecanediamine (60.7 mg;0.276 mmole) in dry pyridine (1 ml). The resulting mixture was stirredat room temperature for 3 hours after which time LCMS showed the absenceof the nitro compound 2 and the presence of product 3 at (M+2H⁺)/2=861.Volatiles were removed in vacuo at 40° C. and the resulting orange oilwas applied to a 10 g Si SPE cartridge eluted with DCM(5×), ether(5×)and EtOAc(5×). The product eluted in the EtOAc fractions as a whitesolid (0.2 g; 22%).

The product may be deprotected using standard techniques.

N.B. The 4-nitrophenylchloroformate should be white with no trace ofyellow colour.

LC/MS Details-Blue Method

Micromass Platform II mass spectrometer operating in positive ionelectrospray mode,

-   mass range 100-1000 amu.-   Column: 3.3 cm×4.6 mm ID, 3 μm ABZ+PLUS-   Flow Rate: 3 ml/min-   Injection Volume: 5 μl-   Solvent A: 95% acetonitrile+0.05% formic acid-   Solvent B: 0.1% formic acid +10 mMolar ammonium acetate-   Gradient: 0% A/0.7 min, 0-100% A/3.5 min, 100% A/1.1 min, 100-0%    A10.2 min    LC/MS Details-Orange Method-   Instrument: Micromass Platform II-   Ionisation Mode: Electrospray +ve-   Range: 100-1000 amu-   Column: 50 mm ×2.1 mm Phenomenex Luna C18, 5 um.-   Flow: 1.0 ml/min-   Inj Vol: 5 ul-   Diode Array Detector: 220-300 nm-   Mobile Phase: A-Water+0.05% v/v TFA.    -   B-Acetonitrile+0.05% v/v TFA-   Gradient: Time % A % B    -   0 100 0    -   8 5 95    -   8.1 100 0

It is to be understood that the present invention covers allcombinations of particular and preferred subgroups describedhereinabove.

Throughout the specification and the claims which follow, unless thecontext requires otherwise, the word ‘comprise’, and variations such as‘comprises’ and ‘comprising’, will be understood to imply the inclusionof a stated integer or step or group of integers but not to theexclusion of any other integer or step or group of integers or steps.

The application of which this description and claims forms part may beused as a basis for priority in respect of any subsequent application.The claims of such subsequent application may be directed to any featureor combination of features described herein. They may take the form ofcomposition, process, or use claims and may include by way of exampleand without limitation the following claims.

1. A compound of formula (I):

wherein R represents a carboxylic acid protecting group; P₁ and P₂ canbe the same or different and are selected from amine protecting groups;P₃ represents a protecting group for 1,2 diols; and LG represents aleaving group.
 2. A compound according to claim 1, wherein R is C₁₋₆alkyl or diphenylmethane.
 3. A compound according to claim 1 or claim 2,wherein P₁ and/or P₂ is/are a t-butoxy carbonyl (Boc) group.
 4. Acompound according to any one of claims 1 to 3, wherein P₃ is CO orCHMe.
 5. A compound according to any one of claims 1 to 4, wherein LG ispara-nitrophenol or a derivative thereof, halide, imidazole orN-hydroxysuccinimide.
 6. A compound according to any one of claims 1 to5, wherein R is methyl or diphenylmethane, P₁ and P₂ are Boc, P₃ is COand LG is para-nitrophenol.
 7. A process for the preparation of acompound of formula (I) as defined in any one of claims 1 to 6, whichcomprises reacting a compound of formula (II);

wherein P₁, P₂, P₃ and R are as defined in any one of claims 1 to 6,with a compound of formula (II);

wherein each LG is as defined in any one of claims 1 to 6, in a solvent,and a base.
 8. A process as claimed in claim 7, wherein the reaction iscarried out in the absence of water.
 9. A process according to claim 7or claim 8, wherein the solvent is pyridine or a pyridine type solvent.10. A process according to any one of claims 7 to 9, wherein the base isa tertiary amine.
 11. A process according to claim 10, wherein thetertiary amine is dimethylaminopyridine (DMAP), 4-pyrrolidinopyridine or1,8-diazabicyclo[5.4.0]undec-7-ene.
 12. Use of a compound of formula (I)as defined in any one of claims 1 to 6, in the preparation of aneuraminidase inhibitor conjugate of formula (V);


13. A process for the preparation of a compound of formula (V) asdefined in claim 12, which comprises reacting a compound of formula (I)as defined in any one of claims 1 to 6, with a compound of formula (VI);

in a solvent and in the presence of base followed if necessary bydeprotection.